EP0639067B1

EP0639067B1 - Use of perfluorcarbons for the preparation of potentiators of the ultrasound induced hyperthermia treatment of biological tissues

Info

Publication number: EP0639067B1
Application number: EP92912400A
Authority: EP
Inventors: Inc. Imarx Therapeutics
Original assignee: ImaRx Therapeutics Inc
Priority date: 1990-09-11
Filing date: 1992-05-04
Publication date: 2002-03-27
Anticipated expiration: 2012-05-04
Also published as: EP0639067A4; AU694973B2; AU1221297A; JPH07506032A; AU678341B2; DE69232518D1; US5149319A; EP0639067A1; DE69232518T2; AU1992692A; WO1993021889A1; ATE214903T1

Abstract

Gas, gaseous precursors and prefluorocarbons are presented as novel potentiators for ultrasonic hyperthermia. The gas, gaseous precursors and perfluorocarbons which may be administered into the vasculature, interstitially or into any body cavity are designed to accumulate in cancerous and diseased tissues. When therapeutic ultrasonic energy is applied to the diseased region heating is increased because of the greater effectiveness of sound energy absorption caused by these agents.

Description

The present invention relates to the use of perfluorocarbons for the preparation of hyperthermia potentiators for use in combination with ultrasound to facilitate the selective heating of the tissues and fluids.
The usefulness of heat to treat various inflammatory and arthritic conditions has long been known. The use of ultrasound to generate such heat for these as well as other therapeutic purposes, such as in, for example, the treatment of tumors has, however, been a fairly recent development.
Where the treatment of inflammation and arthritis is concerned, the use of the ultrasound induced heat serves to increase blood flow to the affected regions, resulting in various beneficial effects. Moreover, when ultrasonic energy is delivered to a tumor, the temperature of the tumorous tissue rises, generally at a higher rate than in normal tissue. As this temperature reaches above about 43°C, the tumorous cells begin to die and, if all goes well, the tumor eventually disappears. Ultrasound induced heat treatment of biological tissues and fluids is known in the art as hyperthermic ultrasound.
The non-invasive nature of the hyperthermia ultrasound technique is one of its benefits. Nonetheless, in employing hyperthermic ultrasound, certain precautions must be taken. Specifically, one must be careful to focus the ultrasound energy on only the areas to be treated, in an attempt to avoid heat-induced damage to the surrounding, non-targeted, tissues. In the treatment of tumors, for example, when temperatures exceeding about 43°C are reached, damage to the surrounding normal tissue is of particular concern. This concern with over heating the non-target tissues thus places limits on the use of hyperthermic ultrasound. Such therapeutic treatments would clearly be more effective and more widely employed if a way of targeting the desired tissues and fluids, and of maximizing the heat generated in those targeted tissues, could be devised.
The present invention is directed toward improving the effectiveness and utility of hyperthermic ultrasound by providing agents capable of promoting the selective heating of targeted tissues and body fluids.
WO93/05819, which forms part of the state of the art by virtue of Article 54(3) EPC, describes gaseous ultrasound contrast media comprising microbubbles of gases that have life spans in solution that are long enough to enable in vivo imaging. The long life spans in solution are achieved by selection of suitable gases for forming the microbubbles.
US-A-4865836 describes the use of emulsions of brominated perfluorocarbon liquids as contrast media in vivo.
WO80/02365 describes microbubbles of gas encapsulated in a suitable membrane for use in diagnostic imaging in vivo.
WO92/22249, which forms part of the state of the art by virtue of Article 54(3) EPC, describes the use of ultrasonic energy to heat biological tissues and fluids, and more specifically, the use of gas filled liposomes prepared by a vacuum drying gas instillation method, and/or gas filled liposomes substantially devoid of liquid in the interior thereof, as hyperthermia potentiators in combination with ultrasound to facilitate the selective heating of the tissues and fluids.
WO91/03267 describes the use of liquid perfluorocarbons to temporarily fill pulmonary air passages and thereby provide a heat transfer medium for hyperthermia treatment of lung cancer. The liquid perfluorocarbon may be heated by ultrasound.
The present invention provides use of perfluorocarbons for the preparation of a product for use in the potentiation of ultrasound induced hyperthermia treatment of biological tissues and fluids, wherein the perfluorocarbon is encapsulated in a liposome.
By using the present invention, hyperthermic ultrasound becomes a better, more selective and more effective therapeutic method for the treatment of tumors, inflammation, and arthritis, as well as other various conditions.
The present invention will now be described in detail with reference to the accompanying drawings, which relate to comparative experiments, as follows:-
Figure 1 provides a graph which plots the temperature over time for three different samples subject to ultrasound treatment using a 1.0 megahertz continuous wave source of ultrasonic energy. Both Sample 1 (multimellar vesicles composed of egg phosphatidylcholine and having encapsulated therein CO₂ gas) and Sample 3 )a phosphate buffered saline solution pressurized with CO₂ gas) have a similar increase in temperature over time. Sample 2 (a degassed phosphate buffered saline solution) exhibited a much lower increase in temperature over time, as compared with Samples 1 and 3.
Figure 2 provides a graph which plots the temperature over time for different samples subjected to ultrasound treatment using a 1.0 megahertz continuous wave source of ultrasonic energy. Sample 2 (a phosphate buffered saline solution pressurized with CO₂ gas) shows a much greater increase in temperature over time than Sample 1 (a degassed phosphate buffered saline solution).
The hyperthermia potentiators employed in the method of the subject invention comprise one or more perfluorocarbons, preferably a perfluorocarbon compound selected from the group consisting of perfluoro- octyliodide, perfluorotributylamine, perfluorotripropyl- amine and perfluorooctlybromide, and any and all combinations thereof. The perfluorocarbons described herein, are encapsulated in liposomes prior to administration.
Liposomes may be prepared using any one or a combination of conventional liposome preparatory techniques. As will be readily apparent to those skilled in the art, such conventional techniques include sonication, chelate dialysis, homogenization, solvent infusion coupled with extrusion, freeze-thaw extrusion, microemulsification, as well as others. These techniques, as well as others, are discussed, for example, in U.S. Patent No. 4,728,578, U.K. Patent Application G.B. 2193095 A, U.S. Patent No. 4,728,575, U.S. Patent No. 4,737,323, International Application WO86/00238 Mayer et al., Biochimica et Biophysica Acta, Vol. 858, pp. 161-168 (1986), Hope et al., Biochimica et Biophysica Acta, Vol. 812, pp. 55-65 (1985), U.S. Patent No. 4,533,254, Mahew et al., Methods In Enzymology, Vol. 149, pp. 64-77 (1987), Mahew et al., Biochimica et Biophysica Acta, Vol. 75, pp. 169-174 (1984), and Cheng et al., Investigative Radiology, Vol. 22, pp. 47-55 (1987), and WO 90/04943. As a preferred technique, a solvent free system similar to that described in International Application WO 86/00238 is employed in preparing the liposome constructions.
The materials which may be utilized in preparing the liposomes of the present invention include any of the materials or combinations thereof known to those skilled in the art as suitable in liposome construction. The lipids used may be of either natural or synthetic origin. Such materials include, but are not limited to, lipids such as cholesterol, cholesterol hemisuccinate, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol, lysolipids, fatty acids, sphingomyelin, glycosphingolipids, glucolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids, polymerizable lipids, and combinations thereof. As one skilled in the art will recognize, the liposomes may be synthesized in the absence or presence of incorporated glycolipid, complex carbohydrate, protein or synthetic polymer, using conventional procedures. The surface of a liposome may also be modified with a polymer, such as, for example, with polyethylene glycol (PEG), using procedures readily apparent to those skilled in the art.
Any species of lipid may be used, with the sole proviso that the lipid or combination of lipids and associated materials incorporated within the lipid matrix should form a bilayer phase under physiologically relevant conditions. As one skilled in the art will recognize, the composition of the liposomes may be altered to modulate the biodistribution and clearance properties of the resulting liposomes.
In addition, the size of the vesicles can be adjusted by a variety of procedures including filtration, sonication, homogenization and similar methods to modulate liposomal biodistribution and clearance. To increase internal aqueous trap volume, the vesicles can be subjected to repeated cycles of freezing and thawing.
The liposomes employed may be of varying sizes, but preferably have a mean outer diameter between about 30 nanometers and about 10 microns. As is known to those skilled in the art, vesicle size influences biodistribution and, therefore, different size vesicles are selected for various purposes. For intravascular use, for example, vesicle size is generally no larger than about 2 microns, and generally no smaller than about 30 nanometers, in mean outer diameter. For non-vascular uses, larger vesicles, e.g., between about 2 and about 10 micron mean outside diameter may be employed, if desired.
The lipids employed may be selected to optimize the particular therapeutic use, minimize toxicity and maximize shelf-life of the product. Neutral vesicles composed of either saturated or unsaturated phosphatidyl- choline, with or without sterol, such as cholesterol, function quite well as intravascular hyperthermia potentiators to entrap gas and perfluorocarbons. To improve uptake by cells such as the reticuloendothelial system (RES), a negatively charged lipid such as phosphatidylglycerol, phosphatidylserine or similar materials is added. For even greater vesicle stability, the liposome can be polymerized using polymerizable lipids, or the surface of the vesicle can be coated with polymers such as polyethylene glycol so as to protect the surface of the vesicle from serum proteins, or gangliosides such as GM1 can be incorporated within the lipid matrix. Vesicles or micelles may also be prepared with attached receptors or antibodies to facilitate their targeting to specific cell types such as tumors.
The perfluorocarbons can be encapsulated by the liposome by being added to the medium in which the liposome is being formed, in accordance with conventional protocol.
The hyperthermic potentiators of the present invention are administered to a biological tissue or to biological fluids, whereupon ultrasound is then applied to the biological matter. The invention is particularly useful when employed in relation to such biological matter as tumor tissue, muscle tissue or blood fluids.
Where the usage is in vivo, administration may be carried out in various fashions, such as intravascularly, intralymphatically, parenterally, subcutaneously, intramuscularly, intraperitoneally, interstitially, hyperbarically or intratumorly using a variety of dosage forms, the particular route of administration and the dosage used being dependent upon the type of therapeutic use sought, and the particular potentiating agent employed. Perfluorocarbons are preferably administered either intravascularly or interstitially. Typically, dosage is initiated at lower levels and increased until the desired temperature increase effect is achieved. In tumors with a principal dominant arterial supply such as the kidney, these hyperthermic potentiating agents may be administered intra-arterially.
For in vivo usage, the patient can be any type of mammal, but most preferably is a human. The method of the invention is particularly useful in the treatment of tumors, various inflammatory conditions, and arthritis, especially in the treatment of tumors. The perfluorocarbons accumulate in tumors, particularly in the brain, because of the leaky capillaries and delayed wash-out from the diseased tissues. Similarly, in other regions of the body where tumor vessels are leaky, the hyperthermic potentiating agents will accumulate.
The potentiators prepared by the invention may be employed in combination with other therapeutic and/or diagnostic agents. In tumor therapy applications, for example, the hyperthermic potentiators may be administered in combination with various chemotherapeutic agents.
Any of the various types of ultrasound imaging devices can be employed, the particular type or model of the device not being critical to the invention. Preferably, however, devices specially designed for administering ultrasonic hyperthermia are preferred. Such devices are described U.S. Patent Nos. 4,620,546, 4,658,828 and 4,586,512.
Although applicant does not intend to be limited to any particular theory of operation, the hyperthermic potentiators prepared by the present invention are believed to possess their excellent results because of the following scientific postulates.
Ultrasonic energy may either be transmitted through a tissue, reflected or absorbed. It is believed that the potentiators prepared by the invention serve to increase the absorption of sound energy within the biological tissues or fluids, which results in increased heating, thereby increasing the therapeutic effectiveness of ultrasonic hyperthermia.
Absorption of sound is believed to be increased in acoustic regions which have a high degree of ultrasonic heterogeneity. Soft tissues and fluids with a higher degree of heterogeneity will absorb sound at a higher rate than tissues or liquids which are more homogeneous acoustically. When sound encounters an interface which has a different acoustic impedance than the surrounding medium, there is believed to be both increased reflection of sound and increased absorption of sound. The degree of absorption of sound is believed to rise as the difference between the acoustic impedances between the two tissues or structures comprising the interface increases.
Intense sonic energy is also believed to cause cavitation and, when cavitation occurs, this in turn is thought to cause intense local heating.
Since perfluorocarbons have high acoustic impedance differences between liquids and soft tissues, as well as decrease the cavitation threshold, the perfluorocarbons may act to increase the rate of absorption of ultrasonic energy and effect a conversion of that energy into local heat.
The potentiators prepared by the present invention may serve to increase the acoustic heterogeneity and generate cavitation nuclei in tumors and tissues thereby acting as a potentiator of heating in ultrasonic hyperthermia. Because the perfluorocarbons create an acoustic impedance mismatch between tissues and adjacent fluids, the perfluorocarbons increase the absorption of sound and conversion of the energy into heat.
The following examples are merely illustrative of the present invention and should not be considered as limiting the scope of the invention as defined in the accompanying claims.
In all of the examples which follow, a 1.0 megahertz continuous wave ultrasonic transducer (Medco Mark IV Sonlator) was used to apply the ultrasonic energy. Degassing of the solution, that is, removal of the gas from the solution, was accomplished by using standard vacuum procedures.

EXAMPLES

Example 1 (comparative)

A cooled degassed solution of phosphate buffered saline (PBS) was subjected to ultrasonic hyperthermia. Another equal volume of standard PBS was pressurized in a commercial soda syphon with carbon dioxide. The pressure was released and the solution was then subjected to ultrasound with identical parameters as for the previously described solution of PBS. The gassed solution reached a significantly higher temperature than the degassed solution. These results are illustrated in Figure 1.

Example 2 (comparative)

Gas bubbles of nitrogen were passed through a standard solution of PBS. A degassed solution of PBS was prepared. Ultrasound energy was applied to each solution, during which time the temperature was measured with a thermometer. The solution containing gas bubbles (Sample 2) reached a significantly higher temperature than the degassed solution (Sample 1). The results in this example are shown in Figure 2, and are qualitatively similar to those observed in Example 1.
In both Examples 1 and 2, it should be noted that the ultrasonic hyperthermia was commenced immediately after gasing the solutions. When ultrasonic hyperthermia was delayed more than five minutes after the gasing step, the resultant temperature was only slightly greater than for the degassed PBS. This is attributed to the relatively rapid decay of the non-stabilized gas bubbles in solution.

Example 3 (comparative)

Liposomes encapsulating gas were prepared via a pressurization process as previously described in WO 91/09629. A liposome without gas was also prepared. The two samples were exposed to ultrasonic energy as described above. The results revealed improved heating for the liposomes that encapsulated the gas similar to that shown in Figure 2. The gas, whether or not entrapped in an outer stabilizing covering such as a liposome, serves to potentiates the heating.
The advantage of using liposomes or other such stabilizing methods is that in vivo the stabilized bubbles may perhaps be more readily directed to sites, e.g., tumors than unencapsulated bubbles. Note that the nonencapsulated bubbles as described in Examples 1 and 2 were only stable for several minutes in solution, whereas the liposomal bubbles will have a much longer stablilty.

Example 4 (comparative)

Albumin microspheres were prepared as previously described U.S. Patent No. 4,718,433 to encapsulate air. Two solutions of PBS were prepared, one containing albumin microspheres encapsulating gas and the other containing a solution of the same concentration of albumin in degassed PBS. The concentration of albumin in both cases was 1%. Ultrasonic energy was then applied as in Example 1. The solution containing the gas filled albumin microspheres reached a significantly higher temperature than the solution of albumin without gas. The temperature increase observed for the gassed solution was similar to that observed for the samples containing gas described in Examples 1 through 3.

Example 5 (comparative)

Stabilized air bubbles were prepared as previously described using a mixture of the polymers polyoxyethylene and polyoxypropylene as in U.S. Patent No. 4,466,442 in solution. Ultrasonic energy was applied. Again, the temperature measurements showed a higher temperature for the solution containing the stabilized air bubbles.

Example 6 (comparative)

A solution containing emulsions of perfluorooctylbromide (PFOB) was prepared as described in U.S. Patent No. 4,865,836 (Sample 1), and the solution was exposed to ultrasonic hyperthermia. Additionally, a second solution of PFOB emulsion was prepared following the same procedures, except that this second solution was gassed with oxygen as described in U.S. Patent No. 4,927,623 (Sample 2). Sample 2 was then exposed to ultrasonic hyperthermia. The Samples 1 and 2 containing the PFOB both achieved a higher temperature upon ultrasound treatment than the degassed PBS of Examples and 2. In addition, Sample 2 reached an even higher temperature with ultrasonic hyperthermia than Sample 1.

Example 7 (comparative)

A tissue equivalent phantom was prepared using low temperature agar gel with a 50°C gelling temperature. A phantom was prepared from degassed PBS and 4% agar gel. Another phantom was prepared, but in this case the liquid gel was pressurized with nitrogen gas at 1.2 MPa (180 psi) for 24 hours in a custom built pressurization chamber at 52°C. The pressure was released over a period of 5 seconds thus forming microbubbles in the liquid yet viscous gel. Both gel samples (degassed and that containing microbubbles) were allowed to gel and to cool to 37°C. The samples were then exposed to ultrasonic energy as above and the temperatures recorded. The sample containing microbubbles again had a much higher rate of heating than the gel prepared from the degassed solution.
The above was repeated but in this case liposomes entrapping gas were placed in the gel and the gel again cooled to 37°C. Ultrasonic heating again showed an improved rate of heating. The purpose of the tissue equivalent phantom was to demonstrate how the bubbles might potentiate heating in tissues, e.g., a tumor.

Example 8 (comparative)

Two rats bearing C2 clonal derived epithelial carcinoma are treated with ultrasonic therapy. In one of these rats, 2 cc of nitrogen gas is injected into approximately 4 cc of tumor volume. Hyperthermia is administered to both rats and the intra-tumoral temperature monitored. The rat treated with an interstitial injection of nitrogen has a higher tumor temperature.

Example 9 (comparative)

One group of rabbits bearing VX2 carcinoma of the brain are treated with ultrasonic hyperthermia while the tumor temperature and the temperature of the surrounding tissue is monitored with a probe. A volume of 3 to 5 cc of perfluorooctybromide emulsion is injected into a second group of rabbits in the carotid artery ipsilateral to the brain tumor, while monitoring the tumor and surrounding tissue. The rabbits treated with the PFOB show increased tumor temperatures and a more selective heating of the brain tumor as compared to the normal tissue.

Example 10 (comparative)

The same experiment as in Example 9 is repeated using a 3 cc injection of liposomes encapsulating gas. Again temperature measurements of tumor and normal tissue show increased temperature in the tumor relative to normal tissue of the animal treated with the gas filled liposomes.

Example 11 (comparative)

A solution of liposomes encapsulating the gaseous precursor methylactate is prepared and suspended in PBS. A control solution of PBS and the solution containing the liposomes encapsulating methylactate is heated with ultrasound and the temperature measured. The temperature of the solution containing the liposomes encapsulating methylactate has a biexponential rate of heating reflecting the improvement in heating efficiency past the point at which gas is formed from the gaseous precursor.

Example 12 (comparative)

In a patient with cancer of the kidney, the left femoral artery is catheterized using standard technique. The renal artery is catheterized and 10 cc of a 1% solution of sonicated albumin microspheres entrapping gas is injected into the renal artery. Therapeutic ultrasound is used to heat the tumor and the microbubbles of gas delivered to the tumor cause improved tumor heating.

Example 13 (comparative)

Example 12 is repeated in another patient but this case gas bubbles encapsulated in the tensides polyoxyethylene and polyoxypropylene are used to embolize the kidney. Again therapeutic ultrasound is applied to the kidney and the result is improved heating of the tumor.

Example 14 (comparative)

Example 13 is repeated but this time using liposomes encapsulating both chemotherapy and carbon dioxide gas. Again hyperthermia is applied to the tumor using ultrasound and not only is there improved tumor heating, but also improved tumor response caused by the interaction of simultaneous heating and chemotherapy.

Example 15 (comparative)

Small liposomes, less than about 100 nm diameter, are prepared to entrap nitrogen gas under pressure. Phase sensitive lipids are selected with gel to liquid crystalline transition temperature of 42.5°C. These are administered intravenously to a patient with glioblastoma multiforme, which is a usually deadly brain tumor. Ultrasonic hyperthermia is applied to the region of the brain tumor through a skull flap which has been previously made surgically. The microbubbles entrapped in the liposomes accumulate in the patient's tumor because of the leakiness of the tumor vessels. The microbubbles are excluded from the normal brain because of the integrity of the blood-brain barrier. The ultrasonic energy raises the tumor temperature to 42.5 degrees centigrade and the liposomes underwent phase transition allowing the bubbles to expand. The intratumoral bubbles increases the effectiveness of heating in the tumor by the therapeutic ultrasound.

Example 16 (comparative)

Air bubbles are entrapped in lipid monolayers as previously described in U.S. Patent No. 4,684,479. In a patient with glioblastoma multiforme, these lipid monolayer stabilized air bubbles are administered I.V. every day for 7 days during daily treatments with ultrasonic hyperthermia. The stabilized air bubbles accumulate in the patient's tumor and the patient has improved response to treatment with ultrasonic hyperthermia.
Various modifications within the scope of the appended claims in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description.

Claims

Use of perfluorocarbons for the preparation of a product for use in the potentiation of ultrasound induced hyperthermia treatment of biological tissues and fluids, wherein the perfluorocarbon is encapsulated in a liposome
Use according to claim 1, wherein the perfluorocarbon is selected from the group consisting of perfluorooctyliodide, perfluorotributylamine, perfluorotripropylamine and perfluorooctylbromide.